Proteolytic enzymes



United States Patent ()7 U.S. Cl. 195-62 18 Claims ABSTRACT OF THEDISCLOSURE Two proteolytic enzymes have been isolated from a species ofmyxobacteria (Sorangiurn), purified and characterized. Both enzymes havebacteriolytic and fibrinolytic properties and exhibit activity againstcertain nematodes and cestodes.

Certain soil bacteria believed to be of the family Myxobacterales and ofthe genus lSorangium have been found to produce two bacteriolyticproteases of different structure and properties. Processes have beendeveloped for the separation and recovery of both enzymes.

Strains of Sorangium have been known to produce extracellular enzymes(see Gillespie and Cook, Can. J. Microbiology 11, 109, 1965). A proteaseand a lysin were described as produced by strains of myxobacteriaincluding Sorangium, particularly strain number 495. The protease hadhydrolytic action on casein and denatured hemoglobin but did not lysebacterial cell walls; and the lysin caused lysis of various species ofbacteria but was claimed to be not a protease.

According to the present invention two distinct proteolytic enzymeshaving lytic activity against bacteria have been isolated from culturefiltrates of Sorangium. The a-enzyme is a serine-protease with the aminoacid sequence -AspSer-Glyat the reactive serine residue. It thus belongsto the same group of serine proteases as all pancreatic serine proteasesof known sequence (e.g. trypsin, chymotrypsin) and not to the groupcharacterized by the sequence ThrSerMet which has been thought toinclude all microbial serine proteases. The B-enzyme cannot be placed inany of the main groups of known proteases.

The myxobacteria Sorangium strains used are maintained in the culturecollection at the Microbiology Research Institute, Canada Department ofAgriculture, Ottawa. The strain designated 495 produces good yields ofthe extracellular enzymes, but other strains such as 18L are alsooperative. The strains of this type producing significant amounts ofextracellular enzymes are operative here.

The organism (Sorangium) may be grown in shake or submerged culture inan aerated aqueous medium contain ing assimilable carbon and nitrogensources and mineral salts. Suitable nitrogen sources include mixtures oforganic and inorganic nitrogen compounds such as acid hydrolyzed caseinand the nitrates. Suitable carbon sources include carbohydrates such asglucose. The mineral salts include phosphates, sulfates, chlorides andmetals such as Fe, Mg, K, Na and traces amounts of zinc. The growthtemperature may range from about 20 to C. with about 25 C. preferred.Enzyme buildup is substantially complete after about to hours althoughsomewhat shorter times may suflice.

The cells are separated after incubation, for example, by centrifugationand the cell-free solution is treated as outlined below to recover thetwo enzymes. It has been found desirable to pass the filtrate through amixed bed of ion exchange resins which have been neutralized pref-Patented June 2, 1970 erably by monovalent acids and bases (e.g. thecation exchanger in ammonium form and the anion exchanger in acetateform) to remove divalent and multivalent ions. The filtrate is acidified(e.g. brought to a pH below about 5 with acetic acid) and contacted atthis pH with a cation exchange resin to adsorb the enzymes. Thistreatment removes practically all bacteriolytic activity; the residualsolution contains substantial amounts of proteolytic activity which canbe recovered by similar adsorption at a lower pH (e.g. about 4). Asuitable cation exchange resin is a weakly acidic carboxylic type suchas Amberlite CG-SO, preferably finely divided and equilibrated withbuffer (e.g. sodium hydroxideacetic acid of pH 5.5 or less and ionicstrength 0.1 or less). If desired (after removing resin from filtrate),both enezymes can be stripped from the resin in one operation e.g. bydispersing the resin in a buffer or salt solution such as 0.2 to 0.4 MNaOH-citric acid buffer of pH about 6.5 to 7.5 adding ammonia or baseuntil the pH of the dispersion is approximately 7. The resulting enzymesolution can be freeze dried without loss of enzyme activity. Thisenzyme mixture could be used in any application where a combined actionof both ocand ,8-enzymes is desirable e.g. animal antihelminthic orWound treatment.

All the proteolytic activity can be removed for the original filtrate byadsorption on the resin at a lower pH (e.g. about 4) and desorbing allactivity, as described above. This recovered enzyme could be used wherethe combined action of all proteolytic enzymes is desirable.

To recover the adsorbed ozand li-enzymes the resin is first washed withdilute buffer such as 0.1 M NaOH- acetic acid pH 5, to removecontaminants without loss of enzyme. The resin is then dispersed inbuffer of ionic strength 0.1 or less and alkali is added until the pH ofthe dispersion is slightly less than neutrality (67). The en zymes canthen be displaced from the resin with more concentrated buifers eitherby gradient displacement (see Example 2) or by stepwise displacement(see Example 3). The B-enzyme is displaced first. The enzymes can berefractionated by similar procedures after readsorption on fresh resin.The final products can be precipitated e.g. with ammonium sulfate, anddialyzed free of salt. The B-enzyme has been crystallized byprecipitation with ammonium sulfate.

In general, the enzymes may be adsorbed from buffer solution of ionicstrength not more than about 0.1 at a pH not greater than about 5. Theresin with adsorbed enzymes can be washed with buffers of similar ionicstrength at pH up to about 5.5 without significant removal of aandfl-enzymes. A partial neutralization of the resin with adsorbed enzymesis desirable using a buffer of low ionic strength but with the pH raisedpreferably to about 6 to 6.5. The enzymes may then be desorbed bybutters or salt solutions of ionic strength greater than 0.1 with a pHat about 5.5 to 6.5 (or above). Preferred butters for desorbing the s-enzymes are of about 0.14 to 0.2 M (in terms of monovalent cation) andof pH 5.5 to 6.5 (more preferably 5.76.2). Buffers for desorbing thea-enzyme are preferably of 0.25 to 0.35 M in monovalent cation (morepreferably 0.25 to 0.3 M) and of the same or slightly increased pH(within the same ranges) as for S -enzyme.

On electrophoresis in tri-s buffer of pH 8 the oz-enzyme migratesslightly faster than egg-white lysozy me, the fl-enzyme slightly slower.The absorptivity (UV) of the OC-EIIZYIIIB at 280 m (millimicrons) wasestimated to be approximately 0.9; that of the fl-enzyme approximately2.0. The B-enzyme has the lower k and the higher absorptivity between260 and 300 mp.

Both the OL- and fi-enzyme-s have molecular weights of approximately20,000. They are both water-soluble basic 3 proteins with completelydifferent amino acid compositions. The following table gives the amountsof several components in each enzyme (see Table 1).

As produced and isolated in this laboratory, the ,8- enzyme contains 1atom of zinc but the zinc can be removed (e.g. by treatment witho-phenanthroline) without loss of lytic activity. The zinc can bereplaced by a divalent metal ion. The a-enzyme is readily inhibited bydiisopropyl phosphorofiuoridate (DFP) but not the fl-enzyme.

Both uand fl-enzymes are proteolytic i.e. both hydrolyze casein, fibrinand the B chain of oxidized insulin. The enzyme has the greaterproteolytic activity towards all three substrates. At pH 9, the a-enzymerapidly cleaves the B chain of oxidized insulin at the linkage betweenamino acid residues 18 and 19, and at the linkages between 12 and 13,and 14-15. Under the same conditions, the fi-enzyme cleaves the B chainrapidly between residues 23 and 24, and more slowly between 18 and 19.The linkages split by both enzymes all involve the carbonyl group of aneutral amino acid. In this respect they resemble pancreatic elastase.Both enzymes solubilize fibrin at physiological pHs (7-8).

Component (residues) a-Enzyme mun- Half cystine. Leucine Tyrosine-Isoleucine. 'lliiol groups Zinc oooolhcm v- Qwwwhcnco Both enzymes havebacteriolytic properties. They hydrolyze linkages of bacterial cell-wallmucopeptides. The fi-enzyme is the more active. Low concentrations(about 1 ug/m1. of the {it-enzyme lyse suspensions of Arthrob acterglobiformis cells completely, and moderately higher concentrations lyseMicrococcus lysodeikticus cells completely within one hour.Corresponding concentrations of the a-enzyrne lyse the suspensionsincompletely, but slightly higher concentrations (about ig/ml.) givecomplete lysis of A. globiformis cells. The bacteriolytic activity doesnot depend upon cleavage of glycosidic linkages in the cell Wallmucopeptide (as is the case with lysozyme) but upon cleavage of amide orpeptide linkages in the cell wall.

The B-enzyme shows strong antinematode activity for example againstAscaris lumbricoides (a parasite of man and pigs), and against larvaloesophagostoma which in the adult form parasitize sheep, while theu-enzyme attacks parasitic cestodes such as the sheep tapeworm Monieziaexpansa (see Examples 9 and 10). Mixtures of the two enzymes would be auseful antinernatode preparation. Neither enzyme has shown toxicity onoral administration to rats in preliminary tests.

By the above procedures, enzyme preparations can be obtained which (a)include all the proteases in the filtrate, (b) mixtures of the 11- andB-enzymes, and (c) the aor fl-enzyme alone. The combination ofproteolytic (and fibrinolytic) activity, bacteriolytic activity,antihelminthic activity plus activity against free-living nematodes,represents usefulness for hydrolysis of various proteins, lysis ofbacteria, debridement of wounds, clearing of clotted fibrin, andcombatting infestations of parasitic helminths and other nematodes. Theenzymes are stable on storage e.g. at 4 C., can be freeze dried and keptfrozen for periods up to at least one year without loss of activity.

The following examples are illustrative of various aspects of theinvention.

4 EXAMPLE 1 Culture filtrate preparation (a) Cultures of Sorangium sp.(isolate No. 495) were maintained on slopes of 0.2% tryptone: 1% agar.After 2 days growth at 25 the cultures were stored at refrigeratortemperatures.

The composition of the culture medium (in grams/ liter) was as follows:Casarnino acids, 10; glucose, 1; K HPO 1; KNO 0.5; MgSO -7H O, 0.2;NaCl, 0.1; FeCI -6H O, 0.01; and NaOH to adjust the pH to 7.0- 7.1 ifnecessary. Trace amounts of zinc were present.

Calcium chloride was omitted from the previously used medium becauseSorangium is less prone to autolysis in the calcium-free medium. Yeastextract was omitted because it interferes with subsequent purificationprocedures.

The organism was grown at 25 in three stages in 2800-ml. Fernbachflasks, containing 500-1000 ml. of medium. This culture provided theinoculum for 12-liter flasks, each containing 6 liters of medium. Thel2-liter flasks, in turn, provided the inoculum for a fermentorcontaining liters of medium. The inocula for the Fernbach flasks weredispersions in sterile water of the growth from a 24-hour slope cultureon culture medium- 1.5% agar. The Fernbach flasks and the 12-literflasks were shaken by a rotary shaker which described a circle of 1 inchdiameter at 113 rpm. The fermentor was of stainless steel constructionand was approximately 2 ft. in internal diameter. The air inlet was a1-inch pipe mounted just above and directed at the stirrer, a 10-inchdisk with eight radial fins, rotating at 580 rpm.

At the end of the growth period, the contents of the ferementor werepassed through a supercentrifuge to remove cells, and the filtratetreated as in Example 2.

(b) The Soranquim organism was grown at 25 in three states ofshake-cultures. Zinc chloride (1 mg./l) was made a component of thegrowth medium as, according to assays of lytic activity, it increasedthe production of ,B-enzyme (a zinc-protein). The first two stages (24hr. each) were in flasks plugged with cotton-wool: 500 ml. flasks with75 ml. of medium, inoculated from a 24 hr. slope-culture, for the firststage and 10.1 flasks with 2 l. of medium, inoculated with 75 ml. of thepreceding shake culture, for the second stage. The glucose wasautoclaved separately When the media for these two stages was prepared.The third stage (48 hr.) was in 40 l. carboys equipped with stoppers andinlet airfilters. Each contained 12 l. of medium. The carboys themseveswere autoclaved, as were concentrated soutions of (a) the glucose, (b)the Casamino acids, (c) zinc chloride, ferric chloride and magnesiumsulfate, and (d) the other mineral salts of the medium @but most of thewater was unsterilized glass-distilled Water. The inoculum was 1 l. ofthe preceding flask stage. Filtered, acid-scrubbed air was passed overthe surface of (not through) the medium. Four carboys were inoculated atone time.

The above procedures require no autoclaving of large volumes of waterand raise no problems with regard to foam-control or pH control.Cultures of Soranquim sp. are less subject to contamination than thoseof most microorganisms but, with only partial sterilization of themedium in the last stage, it is essential that the inoculum be heavy andin vigorous growth. The 48 hr. culture was centrifuged to remove cells.

EXAMPLE 2 The filtrate from Example 1 was contacted with mixedbed ionexchange resin. Amberlite IR45 (acetate) (1100 ml.) and 900ml. ofAmberlite IR120 (NHJ) were added to the liters of filtrate, and thedispersion was left to cool to 5 C. The supernatant solution wasfiltered through 200 ml. of each resin. The resin used for the initialtreatment was drained on the filter and washed with water. The main aimof this step was removal of diand tri-valent ions.

This and all subsequent steps were carried out in a cold room atapproximately 2 C. The filtrate from the previous step was brought to pH4.95 by the addition of 2200 ml. of 20% acetic acid. Two liters (settledvolume) of Amberlite CG50, which had been equilibrated with 0.10 Msodium hydroxide-acetic acid buffer of pH 5.00 was added, with vigorousstirring, in small portions and left to settle overnight. Thesupernatant solution was removed, and the resin was dispersed in 14liters of the above-mentioned acetate buffer and again left to settle.Finally it was washed once more with 9 liters of buffer.

The resin was dispersed in 4 liters of 0.033 M citric acid-sodiumhydroxide buffer of pH 6.25 and the pH of the dispersion was brought to6.25 by capillary inflow, with stirring, of approximately 2 liters of0.5 N sodium hydroxide. The dispersion was added to a large column (12cm. inside diameter) which already contained a 4-cm. bed (about 500 ml.)of Amberlite CG50 that had been equilibrated with similar citratesbuffer. The column was left to drain overnight, the residual supernatantsolution above the resin was removed, and the resin was washed on thecolumn by 2 liters, followed by 0.8 liter, of 0.033 M citric acidbuffer.

One liter of the above 0.033 M citric acid buffer was added to thecolum. A polyethylene inlet plunger was immediately inserted, lockedinto position, and connected to a pump. The subsequent inflow of buffer,extending over days at a flow rate of 90 ml./hour, was as follows. (i)The first 10 liters contained an exponential gradient of sodium citrate.The gradient was generated by three 5- liter vessels connected in lineat their bases; vessel 1 was equipped with a stirrer and connected tothe pump. At the start, vessel 1 contained 5 liters of 0.05 M trisodiumcitrate-citric acid buffer of pH 6.25; vessels 2 and 3 each contained 5liters of 0.10 M trisodium citrate buifer of the same pH. (ii) Thesecond 10 liters had a linear gradient from 0.10 M to 0.15 M trisodiumcitrate-citric acid of pH 6.25. (iii) The final 3 liters was theabovemention 0.15 M buffer.

After this treatment, the resin from the column was dispersed in 3liters of 0.15 M citrate bufler and the pH of the dispersion was broughtto 7.2 with sodium hydroxide. The resulting extract had negligible lyticactivity.

The results of the gradient displacement show lytic activity isdisplaced in two peaks. As shown by the electrophoretic patterns, the,B-enzyme is the major component of the first peak and the wenzyme thatof the second. Differences in A of the ultraviolet absorption spectrumare also evident. [Peak absorptions for or at 280 m, and 5 277 Ill/L].

EXAMPLE 3 The procedures up to and including adsorption of the enzyme onthe resin were unchanged from Example 2. Eight hundred ml. (settledvolume) of Amberlite CG50 adsorbed the enzyme from 4 carboys. Thesettled resin was washed as before with 0.100 M sodium hydroxideaceticacid buffer of pH 5.00.

The major change in the following stages from Example 2 was to replacegradient displacements by stepwise displacements. Gradient generators,pumps, and inlet plungers are not required but, with reliance oncomparatively small hydrostatic pressures to drive buffer through theresin, it is important that the resin be on a support which cannotbecome plugged by the small resin particles. The following steps werecarried out in a cold room at 2 C.

The washed resin, containing enzyme from approximately 100 l. of medium,'was dispersed in 3 l. of cold- .033 M-citric acid-sodium hydroxidebuffer of pH 6.25 and brought to pH 6.4 by capillary inflow of 0.5 Msodium hydroxide (approximately 1800 ml.). The dispersion was added to acolumn which contained one liter of the above mentioned .033 M citratebuffer above a 2 cm. bed of resin which had been equilibrated withsimilar buffer. The resin was left to settle, and the buffer to drain tothe top of the resin bed. The subsequent inputs to the column were:

(a) 4.2 l. of 0.160 M sodium hydroxide-citric acid buffer of pH 5.88;the last 3 l. of the effluent was designated ti-fraction.

(b) 1 l. of 0.210 M sodium hydroxide-citric acid buffer of pH 6.00; thefirst 500 ml. of effluent was added to the ti-fraction; the last 500 ml.was designated a- B-fraction.

(c) 4.5 l. of 0.270 M sodium hydroxide-citric acid buffer of pH 6.20;the eluate was designated or-fraction.

As in Example 2 the ,B-enzyme was eluted first and the OL-BHZYIIIBfollowed. Typical absorbances were of 1.2 for the ,B-fraction and 0.8for the oi-fraction (absorbance per cm. of solution to light of 280nanometer wavelength).

EXAMPLE 4 The ocand fl-fractions from two runs in Example 3 werecombined for refractionation. The first steps were essentially the sameas for the initial fractionations: the solutions were brought to pH 5.0with acetic acid and mixed with a dispersion of Amberlite CG50 inacetate :bufler: approximately 400 ml. (settled volume) of resin for thefi-fraction and 500 ml. for the Ot-fI'ElCtlOH. The resin was washed withacetate buffer, and dispersed in 0.033 M citrate buffer. The dispersionwas brought to pH 6.4 with 0.5 M sodium hydroxide and added to a columncontaining two layers of resin. The lower layer (2 cm.) Was resin whichhad been equilibrated with 0.033 M citric acid-sodium hydroxide bufferof pH 6.25; the upper layer (7 cm.) was resin which had beenequilibrated similarly but, before addition to the column, had beentitrated to pH 6.4 with 0.5 M sodium hydroxide. One liter of the 0.160 Mbuffer, followed by 6 l. of the 0.270 M buffer was used for displacementof the a-enzyme and 3 l. of the 0.033 M bufier followed by 4 l. of the0.160 M buffer was used for displacement of the ,B-enzyme. The enzyme ineflluent with an absorbance greater than about one third of the maximumabsorbance of the peak was then precipitated and dialyzed.

Typical yields of salt-free, freeze-dried enzyme from suchrefractiouations were 44.5 g. of a-enzyme and 2 g. of B-enzyme. Theextent of cross-contamination is extremely low.

EXAMPLE 5 Purification of the tit-enzyme Step 1.-Precz'pitati0n withammonium sulfate and extraction of precipitate.Fractions with eifluentvolumes between 10.3 and 16.5 liters from Example 2 were combined,brought to pH 8.2 with dilute ammonia, brought to roughly 60% saturationwith ammonium sulfate (455 g. of ammonium sulfate per liter ofeffluent), left in an ice bath for 48 hours, and centrifuged for 30minutes at 22,000 g. The supernatant solution was discarded. Thesediment was stirred with about ml. of H 0 to give a turbid solutionwhich was centrifuged for 30 minutes at 78,000 g; the sediment wasreextracted as before with about 10 ml. of water and recentrifuged. Thesupernatant solutions from the two extractions were combined and dilutedwith 30 ml. of 0.230 M sodium hydroxide-citric acid buffer of pH 5.81.

Step 2.-S0lvent exchange in columns of Sephadex G25.The solution ofenzyme from step 1 was added in loads of 3040 ml. to a 2.8 x 66 cm.column of coarseporosity Sephadex G25 which had been washed thorough- 1ywith the above citrate buffer. The enzyme was eluted with the samebuffer at a flow rate of 15 ml./hour. The efliuent with an elutionvolume from to 255 ml. was used in the next step. Apart from exchangingsolvent, this step eliminates a faint yellow color from the enzymesolution.

The recovery of lytic activity in steps 1 and 2 was about 95% Step3.Rechrmat0graplzy on Amberlite CG50.--The upper section of the columnused for this refractionation was of 5.5 cm. inside diameter, the middlesection was 5.7 cm. long and of 2.4 cm. inside diameter, and the lowersection was 4 cm. long and of 1.6 cm. inside diameter. The resin, with abed volume of 130 ml., had been equilibrated with 0.230 M sodiumhydroxide-citric acid buffer of pH 5.81. The gradient was generated by anine-chamber autograd, with 100 ml. of buffer in each compartment. Thebuffers were sodium hydroxide-citric acid buffers of the following pHsand molarities with respect to sodium hydroxide: pH 5.82, 0.230 M(chambers 1, 2 and 3); pH 5.80, 0.263 M (chamber 4); pH 5.75, 0.296 M(chambers 5 and6); pH 5.73, 0.323 M (chamber 7); pH 5.70, 0.350 M(chambers 8 and 9).

For the refractionation to be illustrated, the input to the column wasin 69 ml. of solution from step 2. The enzyme, followed by a fewmilliliters of 0.230 M buffer, was left to flow in under gravity.Fifteen milliliters of 0.230 M buffer was then added to the column, anda polyethylene inlet plunger was inserted. The inflow of buffer from thepump connected to the autograd was 20 ml./hour. Sharp separation ofzit-enzyme was realized. The recovery of lytic units is approximately95%.

Step 4.-Precipitati0n and dialysis.-Enzyme in fractions with eifluentvolumes between 400 and 650 ml. was precipitated with ammonium sulfateas in step 1, dissolved in 0.1 M potassium chloride, and centrifuged asin step 2. It was dialyzed in 3- to 4-ft. lengths of A-inch dialysistubing. The tubing was suspended in glass columns of l-inch insidediameter, through which a slow downward flow of 0.1 M potassium chloridewas passed for 24 hours followed by glass-distilled water for 2 days.The dialyzed enzyme was stored at C.

EXAMPLE 6 Purification of fi-enzyme Steps 1 and 2.Fractions from Example2 with effluent volumes between 3.1 and 7.8 liters were combined andtreated as in steps 1 and 2 of Example 5, with the followingdifferences. (a) The enzyme was precipitated by ammonium sulfate atapproximately 90% saturation. (b) The sediment from the first extractionwith water was dialyzed against 0.1 M KCl to complete the solubilizationof B-enzyme. This point is important, as the precipitated fi-enzymetends to dissolve less readily than its impurities and incompleteextraction reduces enrichment as well as yield. (0) The citrate bufferfor step 2 and for the last stage of step 1 was 0.160 M NaOH-citric acidbuffer of pH 5.80.

Step 3.-Rechr0mat0graphy om Amberlite CG50 by stepwisedispZacement.--The column was as described in step 3 of Example 5. Theresin, with a bed volume of 97 ml., was equilibrated with 0.160 M sodiumhydroxidecitric acid buffer of pH 5.80. The load for the fractionationto be illustrated, 1700 absorbance units in a volume of 2 88 ml., wasadded by gravity flow and followed by 38 ml. of buffer. The inletplunger was inserted, with 50 ml. of 0.160 M buffer above the resin. Thesubsequent inflow of buffer, at ml./hour, was as follows: 400 ml. of0.200 M NaOH-citric acid, 200 ml. of 0.205 M NaOH-citric acid, and 1200ml. of 0.210 M NaOH-citric acid, all of pH 5.77.

Step 4.-Rem0val of trace impurities by precipitation of the enzyme withammonium sulfate.-The enzyme was precipitated at pH 8 by 65% saturationwith ammonium sulfate, left to settle for 24 hours at 0, and centrifugedfor 30 minutes at 50,000Xg. The sediment was stirred for 1 hour at 0with 20 ml. of a 40% saturated solution of ammonium sulfate andrecentrifuged as above. This extraction was repeated twice. Thesupernatant solutions were discarded. The final sediment was then.stirred with 20 ml. of 0.2 M potassium chloride and recentrifuged. Thesupernatant solution, containing 91% of the absorbance units in theoriginal eflluent, was dialyzed for 24 hours against glass-distilledwater by the procedure in step 4 of Example 5. The dialyzed solution wasmixed with one-ninth its volume of 0.200' M potassiumhydroxide-phosphoric acidbuifer of pH 6.5 on 1:10 dilution. The solutionwas brought to 38% saturation by capillary inflow of a saturatedsolution of amomnium sulfate containing a 1:10 dilution of the samepotassium phosphate buffer. The resulting slight precipitate was left tosettle for 24 hours at 0 and re moved by centrifugation for 30 minutesat 50,000 g. The supernatant solution was. then brought to 60%saturation with ammonium sulfate by inflow as above. After 24 hours at0", the precipitated enzyme was removed by centrifugation, dissolved in14 ml. of 0.1 M potassium chloride, and recentrifuged. The supernatantsolution was dialyzed against 0.1 M potassium chloride andglass-distilled water as in Example 5 and stored at 15. The finalrecovery of absorbance units was 70% of the absorbance units in theoriginal effluent. The electrophoretic pattern showed only one componentat loads similar to those tested for the u-enzyrne.

li-Enzyme, prepared as above, crystallized in low yield during adialysis against 0.1 M hydrochloric acid-tris buffer of pH 8.0. It hasbeen crystallized, with high recovery of absorbance and lytic units,from 0.10 M sulfuric acid-tris buffer of pH 8.0, at 10% saturation withammonium sulfate. The crystals were colorless needles with maximumlengths of about 50p.

EXAMPLE 7 The B-enzyme was almost completely freed of zinc by washingthe enzyme in an ultrafiltration cell with 10* M o-phenanthroline in0.01 M acetate buffer of pH 5.5. This treatment had no effect on lyticactivity and did not increase the titer for thiol groups 0.1). Adivalent metal ion was subsequently replaced in the enzyme molecule bydialysis against a solution containing the metal Inhibition by DFP wastested on solutions of enzyme in 0.2 M phosphate buffer of pH 7.7.Treatment of the fi-enzyme for '4 hours with a tenfold excess of DTFPhad no effect on lytic activity; the zinc-free enzyme was equallyinsensitive. Treatment of the OL-CIIZYI'HE with 2.5 mole of DFP per moleof enzyme gave a 95% inhibition of lytic activity within 10 minutes andcomplete inhibition within an hour. The inhibited enzyme was dialysedand then hydrolysed for 20 hours with 2 N HCl at The yield of serinephosphate, 0.375 mole/mole of enzyme is consistent with anesterification by DFP of 1 serine residue of the a-enzyme.

EXAMPLE 8 Substrate:

1 mg. of purified bovine fibrin per 5 ml. of buffer Enzyme oil-4 00 pg.of a-enzyme/S ml.

a2-3 g. of a-enzyme/5 ml.

191-100 g. of B-enzyme/S ml.

,82-3 pg. of fl-enzyme/S ml. Temperature=25.0 C.

Both enzymes are seen to render the fibrin dispersions more clear (or tosolubilize fibrin) with the a-enzyme the more active.

EXAMPLE 98.

Action on parasitic helminths; tests on Ascaris lwmibricoides Testsystem-4 living ascarids in 1 l. beakers containing 100 ml. of 0.9%sodium chloride-0.0025 M/tris buffer and 0.0004 M potassium chloride ofpH 8.7, Temp.=30 C.

Enzymes added at the following levels-(a) 0, (b) 30 ,ug. of oz per ml.,(c) 3 ,ug. of a per ml., ((1) 18 ,ug. of e per ml., (e) 2 ,ug. of 5 perml.

At the end of 24 hrs. incubation on a rotary shaker, all 4 worms inbeakers (a), (b) and (c) were viable and moving actively. Worms in (d)and (e) were dead and their cuticles wrinkled and loose.

The fi-enzyme is shown by tests to have good antinematode activity.

(b) The action of the [El-enzyme on larval Oesophagostomum (from sheepfaeces) was observed under the microscope. After 2 hrs. exposure to anapproximately 0.001% solution of fl-enzyme, the cuticles of the larvaewere noticeably wrinkled and active motion had ceased.

EXAMPLE Test on sheep-tapeworm (Moniezia expansa) In the same buffer asfor Example 9a above, segments of this tape-worm were readilydisintegrated by the OL- enzyme (0.002% wt./ vol. solution) but appearedto be comparatively unaffected by the fi-enzyme (also a 0.002%solution).

I claim:

1. A method for isolating proteases including the aand ,B-bacteriolyticproteases produced by myxobacter Sorangium strain 495 comprising growingthe Sorangium on a medium containing assimilable nitrogen sources,carbon sources and mineral salts including zinc under aerobic conditionsat about 20 to 35 C. until extracellular enzymes have accumulated in themedium, removing the cells, contacting the filtrate with a mixed bed ofneutralized ion exchange resins at about neutral pH, adjusting thetreated solution to a weakly acid pH and contacting with a weakly acidiccation exchange resin at a pH not more than about 5 until theproteolytic enzymes are adsorbed, dispersing the resin with adsorbedenzymes in buffer or salt solution of ionic strength greater than about0.1, adjusting the pH of the dispersion to at least about 5.5 to 6.5,and recovering the desorbed enzymes.

2. The method of claim 1 wherein the cation exchange resin with adsorbedenzymes is washed with butter of ionic strength not more than about 0.1and of pH not greater than about 5.5 to remove impurities other than theaand fi-enzymes, the washed and partially neutralized resin is dispersedin buffer or salt solution of ionic strength greater than 0.1 and the pHraised to at least about 5.5 to 6.5 to desorb the uand ,B-enzymes.

3. The method of claim 2 wherein after removing impurities other thanzxand fi-enzymes from the resin, the aand fi-enzymes are sequentiallydesorbed by a bulfer or salt solution of gradually increasing ionicstrength.

4. The method of claim 3 wherein the ionic strength is increased indistinct steps.

5. The method of claim 2 wherein the aand fi-enzymes are purifiedfurther by readsorption on the cation exchange resin and redesorptionwith selective buffer or salt solutions of increasing ionic strength andpH- 6. The method of claim 2 wherein the Si-enzyme is purified furtherby precipitation from solution with ammonium sulfate and theprecipitated enzyme recovered.

7. The method of claim 2 wherein the enzymes are recovered as salt-freepowders by precipitating with ammonium sulfate, dialyzing theprecipitated enzyme against salt solution and against water, and freezedrying the dialyzed solution.

8. The method of claim 3 wherein buffers with a monovalent cationconcentration of about 0.14 to 0.2 M and pH of about 5.5 to 6.5 are usedto desorb the fi-enzyme selectively, and buffers with a monovalentcation concentration of about 0.25 to 0.35 M and pH of about 5.5 to 6.5are then used to desorb the a-enzyme.

9. The method of claim 1 wherein the cation exchange resin is a finelydivided carboxylic type which has been partially neutralized withmonovalent base.

10. The method of claim 1 wherein the mixed bed includes a weakly basicanion exchange resin in acetate form and a strongly acidic cationexchanger in ammonium form.

11. The method of claim 1 wherein zinc is present in the medium inamounts up to about 1 mg./1.

12. The method of claim 1 'wherein the cells are removed after about40-50 hours growth.

13. The method of claim 1 wherein after the enzymes are adsorbed theresin is washed with low ionic strength acetate buffer of pH about 5,then dispersed in citrate buffer with monovalent cation concentration ofabout 0.1 M and pH about 6, the pH of the dispersion then raised toabout 66.5, and the pand a-enzymes desorbed sequentially by treating theresin with citrate buffer with monovalent cation concentration of 0.15to 0.2 M and pH of 57-62 to desorb the fl-enzyme, followed by the samebuffer of 025-03 M and increased pH within the range 5.7-6.2 to desorbthe a-enzyme.

14. Alpha bacteriolytic serine protease, an extracellular metabolite ofmyxobacter Sorangium strain 495 having high enzyme production,characterized by molecular weight about 20,000, an amino acid sequenceat the active serine site of Asp-SerGly-, lysing various bacteria bycleaving amide or peptide linkages, solubilizing fibrin at pH 6-8,having an absorptivity at 280 millimicrons of approximately 0.9, beingreadily inhibited by diisopropyl phosphorofluoridate, containing interalia the following residues or components (moles per mole of enzyme):histidine 1, lysine 2, arginine 12, leucine 10, isoleucine 8, tyrosine4, with no free thiol groups, and being active against parasiticcestodes.

15. Beta bacteriolytic protease, an extracellular metabelite ofmyxobacter Sorangium strain 495 having high enzyme production,characterized by molecular weight about 20,000, being not inhibited bydiisopropyl phosphorofiuoridate, crystallizing in acicular form,containing one atom of zinc per molecule, lysing various bacteria bycleaving amide or peptide linkages, solubilizing fibrin at pH 6-8,having an absorptivity at 280 millimicrons of about 2, containing interalia the following components in moles per mole enzyme: histidine 8,lysine 3, argine 5, leucine 9, isoleucine 4, tyrosine 13, with no freethiol groups, and being active against nematodes.

16. The beta enzyme of claim 15 wherein the atom of zinc has beenremoved.

17. The beta enzyme of claim 15 wherein the zinc has been replaced by adivalent metal ion.

18. An enzyme mixture consisting essentially of alphaandbeta-bacteriolytic proteases, extracellular metabolites of myxobacterSorangium strain 495.

References Cited Gillespie, D. C., et al.: Canadian Journal ofMicrobiology, vol. 11, No. 1, February 1965 pp. 109-118.

Dixon et al.: Enzymes, 2nd ed., 1964, pp. 26-36 and 41-44.

LIONEL M. SHAPIRO, Primary Examiner U.S. Cl. X.R. --66; 42494

